Prostaglandin (PG), as well as thromboxane, is a physiologically active substance known as “prostanoid,” and it is a lipid having a prostanoic acid skeleton. Prostanoid such as prostaglandin is biosynthesized from arachidonic acid that is released from a membrane phospholipid by the action of phospholipase A2. Prostaglandin is classified into groups A to J, based on differences in the types of an oxygen atom attached to the 5-membered ring thereof and a double bond. In addition, prostaglandin is classified into groups 1 to 3, based on the number of double bonds on the side chain of the prostanoic acid skeleton. For instance, prostaglandin E (PGE) includes PGE1, PGE2 and PGE3, which are different from one another in terms of the number of double bonds on the prostanoic acid skeleton side chain.
Regarding PG, PGH2 is generated from PGG2 that is biosynthesized from arachidonic acid by the action of cyclooxygenase I (COX-I) or cyclooxygenase II (COX-II), and then, PGD2, PGE2, PGF2α and the like are generated based on a difference in the cleavage of the bond between oxygen atoms. Thereafter, PGA2, PGC2 and the like are generated from PGE2. Each PG generation reaction occurs by the action of a specific enzyme, and it is considered that such enzyme has tissue specificity and generates PG suitable for the function of each tissue.
Among various PGs, it is considered that PGE plays various important biological activities and that, through the mediation of its specific receptor, PGE is associated with regulation of the immune system, as well as vasodilatation, a decrease in blood pressure and uterine contraction. The PGE2 receptor is a seven transmembrane G-protein-coupled receptor, as with other PG receptors. The PGE2 receptor is abbreviated as EP, and it was revealed that EP has 4 types of subtypes (EP1, EP2, EP3 and EP4). Each subtype is associated with various phenomena in vivo. That is. EP1 is associated with an increase in intracellular Ca2+ concentration; EP2 and EP4 are associated with an increase in cAMP level; and EP3 is associated with a decrease in cAMP level (Non Patent Literature 1). The 4 types of subtypes have high homology to one another in terms of protein structure.
It has been reported that when a low-molecular-weight compound antagonist having high selectivity to EP4 is administered to mice that had been induced to have experimental autoallergic encephalomyelitis or contact hypersensitivity, accumulation of both TH1 and TH17 cells in the regional lymph node is reduced, and the progression of the disease is suppressed (Non Patent Literature 2). It has been demonstrated that PGE2 promotes production of IL-23 in dendritic cells, as a result of an increase in cAMP level mediated by the activation of EP4. In addition, it has also been demonstrated that, in TH17 cells, PGE2 is involved in proliferation of the TH17 cells in coordination with IL-23. Thus, it has been demonstrated that an increase in cAMP level mediated by the activation of EP4 plays an important role for intracellular signaling in TH17 cells (Non Patent Literature 3). These reports suggest that the PGE2 receptor antagonist, in particular, an EP4-selective antagonist be effective for the treatment of diseases caused by immunological abnormality, with which TH1 or TH17 is associated, such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease and contact dermatitis (Non Patent Literature 2).
It has been reported that many types of cancer cells overexpress COX-II when compared with normal cells. Moreover, it has also been reported that PGE2 acts on cancer tissues or tissues around the cancer tissues and is involved in the progression of cancer. For example, it has been described that PGE2 is involved in infiltration of refractory inflammatory breast cancer cells or lung cancer cells into a metastatic tissue (Non Patent Literatures 4 and 5). Furthermore, it has been known that PGE2 is associated with proliferation of non-small cell lung cancer cells, colon cancer cells, inflammatory breast cancer cells, B lymphocytes, prostatic cancer cells and melanoma, via EP4.
PGE2 has been known to inhibit the function of NK cells which have an action to directly attack cancer cells. One of the mechanisms of PGE2 to inhibit the activity of NK cells is an increase in intracellular cAMP level mediated by the activation of EP4 (Non Patent Literature 6). It has also been known that Treg cells that possibly suppress immunity to cancer are activated via EP4, and the possibility of decreasing the immune system to cancer cells in vivo has been suggested (Non Patent Literature 7). According to these reports, it is apparent that PGE2 is important for the progression of cancer. Hence, clinical studies have been conducted using non-selective inhibitors to COX involved ingeneration of PGE2. However, sufficient therapeutic results could not be obtained due to the side effects of the inhibitors. The PGE2 receptor antagonist, in particular, an EP4-selective antagonist directly suppresses proliferation of cancer cells and boosts the host immune system to cancer. Accordingly, it is anticipated that an antibody that selectively binds to the EP4 receptor will be effective for the treatment of various types of cancers such as breast cancer, colon cancer, lung cancer, prostatic cancer, skin cancer and B-lymphoma.
Conventionally, non-specific COX inhibitors have been applied for the relief of pain. However, it has been known that such non-specific inhibitors cause side effects such as heartburn, indigestion, nausea, abdominal distension, diarrhea, gastralgia, peptic ulcer or gastrointestinal bleeding. In recent years, COX-II selective inhibitors (e.g. celecoxib and rofecoxib) have been developed for the purpose of treating pain. However, it has been suggested that such COX-II selective inhibitors develop severe cardiovascular disorder in specific patients, and thus it has been desired to develop a drug for relieving pain based on a different mode of action. Among PGs generated by COX, PGE2 is known to enhance the hypersensitivity of pain sense. It has been demonstrated by multiple animal experiments that, among PGE2 receptors, EP4 is particularly associated with the enhancement of the hypersensitivity of pain sense. For example, it has been known that the expression of EP4 is enhanced in the dorsal root ganglion (GRG) in a rat model of inflammatory pain, and that a comparatively selective EP4 antagonist (AH23848) relieves the sensitivity of pain in the aforementioned model (Non Patent Literature 8). Moreover, in an analysis using EP4 knock-out mice as well, the same results could be obtained (Non Patent Literature 9). These reports suggest that a pharmaceutical product for selectively blocking the function of EP4 be effective for the treatment of diseases associated with immunological abnormality, cancer and pain, while having fewer side effects.
As methods for selectively blocking the function of EP4, several low-molecular-weight compound antagonists have been reported. However, none of such compound antagonists have been successful as pharmaceutical products. Such low-molecular-weight compound antagonists would be improved in terms of binding selectivity to PGE2 receptor subtypes (EP1-4) or alleviation of binding affinity for thromboxane or other prostanoid receptors. It is concerned that the same side effects as those of COX inhibitors are generated unless sufficient receptor selectivity is secured.
An antibody selectively binding to the EP4 receptor is expected to have higher selectivity than low-molecular-weight compounds. Furthermore, since an antibody drug generally has a longer half-life in blood than low-molecular-weight compounds, it is expected to have drug effects for a long period of time by a single administration. Such antibody drug is effective for chronic diseases (e.g. rheumatoid arthritis, colitis, cancer, etc.).
In general, as a main action mechanism of an antibody drug directed at a membrane protein (receptor), the antibody has recognized cells expressing the protein, and has then removed the cells based on complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). However, since CDC or ADCC is associated with activation of inflammatory cells such as macrophage, such antibody drug is not necessarily suitable for the treatment of diseases caused by immunological abnormality or pain. Accordingly, when a monoclonal antibody capable of selectively inhibiting EP4 is applied for the treatment of diseases caused by immunological abnormality or pain, it is desirably a functional antibody that depends on neither CDC nor ADCC. That is to say, an antibody for selectively blocking EP4-dependent intracellular signaling is desirable.
To date, Japanese Patent No. 3118460 (Patent Literature 1) discloses a method for obtaining an antibody against EP4. However, there have not yet been any reports regarding a specific antibody that EP4-specifically suppresses the function of EP4 at a low dose and binds to neither EP1, EP2 nor EP3. In addition, it has been known that it is difficult to obtain a functional antibody against a seven-transmembrane receptor by the general method for obtaining a monoclonal antibody described in Japanese Patent No. 3118460.